Friday, 20 March 2015

Oxford Nanopore MinION for ctDNA sequencing


A great poster at AGBT was presented by Boreal Genomics and available on the Nanopore wiki for MAPpers. In A nanopore liquid biopsy Patrick Davies describes their combination of the Boreal On-Target with ONT MinION sequencing to detect mutant allele fractions in ctDNA of sub 0.1%. I spoke briefly to Andre Marziali (Boreal Founder & CSO) about the work and summarise the poster here.

In the poster they describe the construction of a test sample made by mixing control plasma DNA and plasma DNA from a colorectal cancer patient (see their 2014 paper) with a KRAS G13D mutation; the test sample had a 0.03% mutant allele frequency. This patient sample had previously been sequenced on MiSeq. Plasma DNA was extracted using extracted DNA using Qiagen’s Circulating Nucleic Acid kit. Mutant alleles were On-Target enriched and sequenced on MinION as part of the MAP.

Boreal’s technology: The On-Target system (PLoS 2012, and in Oncotarget 2014) enriches for mutant alleles by physically separating the mutant alleles from wild-type using SCODA (Synchronous Coefficient of Drag Alteration). The region of interest is first PCR amplified and this PCR product is injected into a cartridge containing acrylamide gel-bound mutant allele oligos (in this case a mix of 8 KRAS mutant alleles including G13D). An oscillating electric field and temperature variation around Tm are used to capture and enrich mutant DNA from wild-type. Multiple cycles move the mutant DNA to an extraction well in the system whilst wild-type DNA is pushed in the opposite direction. They have a nice video on their website.


MinION sequencing: the mutant DNA fragments from the On-Target system are PCR amplified before ONT library prep: end repair, A-tailing and adapter ligation (sounds familiar); and sequencing on the MinION. The poster figures describe how  “clinically relevant ctDNA detection is limited by the sequencing error rate of the MinION” but that after On-Target enrichment “WT DNA [is removed] from the sample, [and] sequencing errors no longer generate false positive signals, improving specificity”. The MinION reported no WT reads from the normal samples after On-Target, but detected 0.23% and 0.01% KRAS G13D in the patient DNA and the titration respectively; where 0.3% and 0.03% were expected.

The numbers of reads called as WT or mutant KRAS were reported in the poster, and although the numbers are very low (perhaps too low) there is a clear enrichment of KRAS G13D over other KRAS mutant alleles (included in the On-Target chip). In the patient 460 reads included 84 G13D plus 14 other KRAS mutant alleles, and 2 WT KRAS reads. In the titration 360 reads included just 3 G13D plus 3 other KRAS mutant alleles, and 0 WT KRAS reads. The normal control had 353 reads with no KRAS alleles mutant or wild-type. It is unclear to me from the poster how Boreal calculate the expected allele frequency using their COGS control amplicon. However the very small numbers of reads, whilst demonstrating that this method is possible, mean the results are likely to be  quite variable from prep to prep and run to run. I would worry about sampling error affecting results at such low depth.

Is MinION a good tool for ctDNA: The biggest strengths of MinION are  most obviously (IMHO) the length of reads and the speed; and hopefully base-modifications in the future. ctDNA is only 170bp in length and PCRs from ctDNA are usually shorter so this application does not make use of the MinION potential read-length. However ctDNA applications are likely to benefit from being run fast. At AHSG Oxford Nanopore demonstrated real-time analysis of a mix of bacterial genomes, so this might enable not just fast sequencing but also fast reporting of ctDNA results.


Is a nanopore liquid biopsy possible in a GP surgery: An exciting possibility of the MinION is its extreme portability due to the small size – I accidentally took mine home after a meeting because it was in my pocket (try saying that about any other sequencer)! The other exciting possibility was direct sequencing of DNA from blood (as proposed by Clive Brown at ONT’s launch in AGBT’12). Can the two be brought together to confirm the presence of mutant alleles while you wait or after your appointment?

Imagine combining MinION real-time analysis with the Tweet alerts available from minoTour (Nottingham DeepSeq Informatics Team); a Tweet alert to you and your GP that says no TP53 mutations found?

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